Utilization of heme-hemopexin as a source of heme by Haemophilus influenzae type b is dependent on expression by this bacterium of the 100-kDa HxuA protein, which is both present on the bacterial cell surface and released into the culture supernatant (L. D. Cope, R. Yogev, U. Muller-Eberhard, and E. J. Hansen, J. Bacteriol. 177:2644-2653, 1995). Radioimmunoprecipitation analysis showed that the soluble HxuA protein present in H. influenzae type b culture supernatant bound heme-hemopexin complexes in solution. An isogenic H. influenzae type b hxuA mutant was unable to utilize soluble heme-hemopexin complexes for growth in vitro unless soluble HxuA protein was provided exogenously. Soluble HxuA protein secreted by a nontypeable H. influenzae strain also allowed growth of this H. influenzae type b hxuA mutant. These results indicated that the heme present in heme-hemopexin complexes is rendered accessible to H. influenzae when these complexes are bound by the soluble HxuA protein.